CD39 is a cell-surface antigen that was originally identified as a marker for mature B cells, but is also expressed on less mature B cells, Epstein-Barr Virus-transformed B cells, activated T cells, endothelial cells and some myeloid cell lines (Dörken et al., in Leukocyte Typing IV; W. Knapp, B. Dörken, and W. R. Gilks, Eds; Oxford University Press, New York, N.Y.; pp. 89-90, 1989). Monoclonal antibodies against CD39 induce B cell homotypic adhesion, an activity that may be important in the regulation of immune function (Kansas and Tedder, J. Immunol. 147:4094-4102, 1991). Molecular cloning and characterization of CD39 indicated that it is unique cell surface molecule that contains two potential transmembrane regions and a hydrophobic segment within the putative extracellular domain (Maliszewski et al., J. Immunol. 153:3574, 1994). The amino acid sequence of CD39 was reported to exhibit some homology with a guanosine diphosphatase from yeast (Maliszewski et al., supra).
In 1996, an ATP diphosphohydrolase was cloned from potato tubers (Handa and Guidotti, Biochem. Biophys. Res. Commun. 218:916, 1996). The amino acid sequences of this and several other NTPases demonstrated a high degree of similarity, particularly within several small “apyrase conserved regions” (ACR). CD39 shares these conserved regions with soluble ATP-diphosphorylase from potato tubers, other apyrases and related enzymes. It was subsequently reported that native and recombinant full-length CD39 possess E-type ATP diphosphohydrolase (ATPDase) activity (Marcus et al., J. Clin. Invest. 99:1351, 1997); Kaczmarek et al., J. Biol. Chem. 271:33116, 1996); Wang and Guidotti, J. Biol. Chem. 271:9898, 1996). ATPDases degrade nucleoside tri- and/or diphosphates, but not monophosphates (Plesner, Int. Rev. Cytol. 158:141, 1995).
Vascular endothelial cells constituitively express a cell-surface ADPase (ecto-ATP diphosphohydrolase, apyrase, EC 3.6.1.5), one of at least 3 thromboregulatory systems which function in the maintenance of blood fluidity (Marcus and Safier, FASEB J. 7:516, 1983; Marcus et al., J. Clin. Invest. 88:1690, 1991). This ecto-ADPase, which belongs to the E-type ATPDase family, rapidly metabolizes ADP in the platelet releasate, terminating further platelet recruitment and aggregation.
Immunoprecipitation of HUVEC detergent lysates with anti-CD39 mAb resulted in complete capture of cell-associated ADPase activity, suggesting that CD39 is the only ecto-ADPase on endothelial cells (Marcus et al., J. Clin. Invest. 99:1351, 1997). In the same study, COS cell transfectants expressing recombinant CD39 at the cell surface totally inhibited ADP-induced platelet aggregation. Thus, CD39 plays a prominent role in thromboregulation (see also, Gayle et al., J. Clin. Invest., 101:1851, 1998; WO96/30532).
Excessive platelet activation (i.e., stimulation by an agonist) and recruitment, leading to platelet aggression and vessel occlusion at sites of vascular injury in the coronary, carotid, and peripheral arteries, presents a major therapeutic challenge in cardiovascular medicine. Excessive platelet activation and recruitment is a contributing factor in clinical disorders including stroke, unstable angina, myocardial infarction, and restenosis following percutaneous coronary intervention including angioplasty, atherectomy, stent placement, and bypass surgery.
Glycoprotein IIb/IIIa an antagonists, such as the monoclonal antibody marketed as ReoPro® (Centocor Inc.), are presently under development for the inhibition of platelet aggregation in patients undergoing percutaneous coronary intervention, and in patients with acute coronary syndromes such as unstable angina and myocardial infarction. The activation glycoprotein IIb/IIIa receptors, however, is a late event in the cascade that leads to platelet aggregation.
There is a great need to identify additional therapeutic strategies and compositions for the pharmacological neutralization of platelet reactivity (activation, recruitment, aggregation). In particular, there is a need to identify compounds and compositions which target early portions of coagulation pathways such as the ADP-dependent activation and recruitment of platelets. There is, in fact, an urgent need to identify new strategies and compositions for the treatment of stroke, which is the third leading cause of death in the United States. In the case of stroke, an advantageous therapeutic agent will reduce intravascular thrombus burden and accompanying neurological defects without increasing intracerebral hemorrhage.